CN102911868A - Microorganism culture medium and microorganism culture method - Google Patents
Microorganism culture medium and microorganism culture method Download PDFInfo
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- CN102911868A CN102911868A CN201210369787XA CN201210369787A CN102911868A CN 102911868 A CN102911868 A CN 102911868A CN 201210369787X A CN201210369787X A CN 201210369787XA CN 201210369787 A CN201210369787 A CN 201210369787A CN 102911868 A CN102911868 A CN 102911868A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6409—Fatty acids
- C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention discloses a microorganism culture medium and a microorganism culture method. The culture medium comprises a combination of strongly acidic and weakly alkaline salt and weakly acidic and strongly alkaline salt, accordingly has certain pH (potential of hydrogen) control ability, and can control the pH within a constant and suitable range in a culture process, operational steps for supplementing acid and alkali by external equipment to adjust the fermentation pH are reduced or omitted, potential hazards of contamination caused by the operational steps are also reduced or omitted, and the controllability and the stability of a fermentation process are greatly improved. Besides, ammonium salt used as the strongly acidic and weakly alkaline salt can also be used as a nitrogen source to replace parts of yeast extracts, so that consumption of expensive nitrogen sources is reduced, and the cost of the culture medium can be greatly lowered.
Description
Technical field
The invention belongs to microorganism field, relate in particular to a kind of microbiological culture media and cultural method.
Background technology
Polyunsaturated fatty acid (polyunsaturated fatty acids PUFA) refers to contain two or more pairs key, and carbon chain length is the straight chain fatty acid of 16～22 carbon atoms, it is the biomembranous important composition composition of cell and organism, adjustable ganglion cell's configuration, running balance, keep the relatively mobile of cytolemma, to keep the normal physiological function of cell, therefore can affect the chemical constitution of cell, signal transmits, the functions such as immunity, thereby great with being related of relative disease, PUFA has important physiological regulation function in human body, comprise and make the cholesterol esterification, reduce blood cholesterol and triglyceride level, blood viscosity lowering improves blood microcirculation, improve the activity of brain cell, memory and thinking ability etc.Polyunsaturated fatty acid mainly comprises timnodonic acid (EPA), docosahexenoic acid (DHA) etc., DHA has prevention and Cardiovarscular, improve blood viscosity and improve erythrocyte deformability, prevention and treatment cancer, antithrombotic, the effects such as anti-inflammatory, DHA is one of chief component material of human brain, the normal development of baby's brain and the normal performance of adult's brain function there is very important effect, the shortage that studies have shown that DHA can cause brain function to reduce, so expert advice grownup and pregnant woman and the food that need to additional be rich in PUFA, replenish in order to an amount of such as ocean fish etc., DHA is well medicine and nutritive food composition.
In view of critical function and the effect of PUFA, extensively receive the concern of medical profession and food circle at present.The commercial source of tradition preparation PUFA is to separate from fish oil; but from fish oil, extract PUFA and contain strong fishy smell; and resource-constrained; output is unstable; the content of W-3 polyunsaturated fatty acid is because of the kind of fish in the fish oil; the season of fishing for; weather and place different and exist difference; the fish oil output fluctuation is very large; and purifying process is complicated; the product yield is low; some excessive appearance of fishing for behavior of ignoring the possible consequences for commercial benefits that the while product consumption causes greatly; brought disadvantageous effect to environmental resource protection, therefore traditional fish oil preparation method can not satisfy the demand of society.
Found at present to utilize microorganism to produce PUFA; and existing many research reports; particularly utilize the polyunsaturated fatty acids such as algae fermentative Production DHA to become present scholar's study hotspot; the method is not subjected to season; the condition restriction such as region; fatty acid content height and component are single; greatly simplify purifying process and reduced refining difficulty; the culture condition ratio is easier to control; and then for control lipid content and composition provide may; utilize the polyunsaturated fatty acids such as DHA Production by Microorganism Fermentation can also reduce because of the market requirement and fish in a large number the impact on environmental resources that brings, the protection of environmental resources is also had great importance.
The Production by Microorganism Fermentation polyunsaturated fatty acid can be realized culture condition, controls such as the operation of temperature, dissolved oxygen, pH etc., thus controllability and the stability of easier realization culturing process and product quality.The problem that often occurs during the fermentation is, because appearance and/or certain medium component of meta-bolites are consumed, violent fluctuation can appear in pH in the fermentation culture process, under these circumstances, if and in fermentation process, pH is not carried out suitable regulation and control, cellular metabolism speed and upgrowth situation will be affected, even stop growing, therefore, the fermentation culture of microorganism need to be regulated pH.
At present in the achievement in research, microbial fermentation is produced polyunsaturated fatty acid fermenting process pH regulate and control method and is commonly and adopts peripheral equipment supplemental acid alkali lye in the fermentation system to regulate, though this method can be controlled at constant scope with fermentation pH to a certain extent, but also there is some problems, for example increase the fermenting process equipment investment, sterilization process and artificial energy consumption, may bring the hidden danger that other microorganisms cause the fermenting process microbiological contamination into and increased most serious of all, in microbial fermentation is cultivated, in case microbiological contamination will cause tremendous influence and financial loss to the product stably manufactured.
Except adopting peripheral equipment, in the last few years, himself possessed certain pH regulator ability and also become and in the microbial fermentation pH is regulated and control a research direction and effective means by medium component adjustment is made.These methods mainly can be divided into following a few class:
(1) in substratum, adds strong base-weak acid salt.CN101538592B discloses a kind of method of producing docosahexenoic acid with Kou Shi Crypthecodinium cohnii industrial fermentation, and it adds Sodium Glutamate in substratum, do not regulate during the fermentation pH; CN101519676B discloses a kind of method of producing docosahexenoic acid with Kou Shi Crypthecodinium cohnii industrial fermentation, and it adds Sodium Glutamate in substratum, and also replenishes during the fermentation citric acid to regulate pH; CN1218035C discloses a kind of marine microalgae heterotrophism that utilizes and has cultivated the production long chain polyunsaturated fatty acids, and it adds Sodium Glutamate in substratum, do not regulate during the fermentation pH; CN101591617B discloses a kind of docosahexaenoic acid-producing strain and mutagenesis screening method and its application, and it adds Sodium Glutamate and sodium bicarbonate in substratum, do not regulate during the fermentation pH.Nitrogenous source adds in the method for the strong base-weak acid salts such as Sodium Glutamate in these employing substratum, strong base-weak acid salt causes fermenting process pH to occur significantly rising after by cell consumption, and pH can have a strong impact on and change cellular process when too high, and then cause the fermenting process underproduction, and when the needs peripheral equipment replenishes acid solution adjusting pH, not only increase equipment investment, also have simultaneously the microbiological contamination risk.
(2) in substratum, add strong acid weak base salts such as adding inorganic ammonium salt.CN 101528939B discloses and has used the substratum of improvement to produce omega-fatty acid from the thraustochytriale flora, adds during the fermentation sodium hydroxide and regulates pH; CN101812484A discloses a kind of method of producing DHA by Schizochytrium in high-density culture through fermentation, replenishes during the fermentation ammoniacal liquor and regulates pH; A series of patents of the people such as Richard B.Bailey disclose respectively by cultivating the method that eukaryotic microorganisms increases the generation of the lipid that contains polyene fatty acid at the fermentor tank middle-high density, during the fermentation, replenish ammoniacal liquor and regulate pH.Nitrogenous source adds in the method for the strong acid weak base salts such as inorganic ammonium salt in these employing substratum, causes fermenting process pH to occur significantly descending after the strong acid weak base salt consumption.PH crosses when hanging down and will cause fermentation system to become sour, and can have a strong impact on and change cellular process, and then cause the fermenting process underproduction, and strong acid environment is serious to the fermentation equipment infringement simultaneously, is unfavorable for utilizing the extensive and steady in a long-term unsaturated fatty acids of producing of microorganism.If add during the fermentation alkali lye or other high pH solutions are regulated, then increased equipment investment, and had the microbiological contamination risk.
In addition, CN1914327A discloses a kind of method of the THRAUSTOCHYTRIALES of cultivation microorganism belonging to genus, and it adopts in substratum and to add calcium carbonate and regulate and control and stable fermenting process pH.But because the carbonic acid gas that calcium carbonate forms during the fermentation only has limited solubleness in water, thereby cause the during the fermentation reduction of surge capability of this buffer system.
Therefore this area needs new substratum scheme to realize the efficient stable control of pH in the fermenting process.
Summary of the invention
The invention provides a kind of microbiological culture media scheme; fermentation culture process pH raises or the impact of the fluctuation cell growth that descends in order to solve; reduce or eliminate the fermentation culture process and carried out the operation of acid-alkali accommodation by peripheral equipment; and the fermenting process microbiological contamination hidden danger of bringing thus; this substratum scheme is controlled at culturing process pH in the constant scope by the combination of nutritive substance; realize the easy and efficient regulation and control of fermentation culture process pH, significant to the large-scale production of microbial fermentation production unsaturated fatty acids.
For reaching this purpose, the present invention by the following technical solutions:
In first aspect, the invention provides a kind of substratum, comprise the combination of strong acid weak base salt and weak acid strong alkali salt in the described substratum to control during the fermentation pH, wherein, described strong acid weak base salt is organic and the inorganic acid ammonium salt, is preferably ammonium nitrate or ammonium sulfate, ammonium chloride, ammonium oxalate more preferably is ammonium sulfate, and described weak acid strong alkali salt is carbonic acid or amino acid whose sodium salt, sylvite or calcium salt.
In substratum of the present invention, the combination of described strong acid weak base salt and weak acid strong alkali salt can be used for the pH of described substratum is controlled to be 4.0-9.0, preferably 5.0-8.0, most preferably 6.0-7.0.
In substratum of the present invention, described amino acid whose sodium salt, sylvite or calcium salt can be sodium salt, sylvite or calcium salt or its mixtures of at least 2 kinds of L-glutamic acid, aspartic acid, Methionin, arginine, Histidine, glycine, Serine, Threonine, halfcystine, tyrosine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, tryptophane or methionine(Met).
In substratum of the present invention, described weak acid strong alkali salt can be Sodium Glutamate.
In substratum of the present invention, the content of Sodium Glutamate can be 1-20g/L, is preferably 5-15g/L, and more preferably 8-12g/L most preferably is 10g/L.
In substratum of the present invention, the content of ammonium sulfate can be 0.5-5g/L, is preferably 1-4g/L, and more preferably 2-3.5g/L most preferably is 3g/L.
Substratum of the present invention can also comprise other nitrogenous sources, carbon source, inorganic salt and trace element;
Preferably, described carbon source is waste molasses, sugar cane juice, Semen Maydis powder, sucrose, fructose, glucose, Zulkovsky starch, carbonic acid gas or its mixture of at least 2 kinds;
Preferably, described nitrogenous source is organic nitrogen compound, inorganic nitrogen compound or its mixture;
Preferably, described inorganic salt comprise sodium salt, magnesium salts, sylvite, molysite, calcium salt, vitriol, carbonate, supercarbonate, acetate or its mixture of at least 2 kinds; And/or,
Preferably, described trace element comprises VITMAIN B1, vitamin B6, vitamin B12, vitamin H, 6-benzyl aminoadenine (6-BA) or its mixture of at least 2 kinds.
In substratum of the present invention, described organic nitrogen compound comprises yeast extract, yeast extract paste, corn steep liquor, peptone, amino acid, Sodium Glutamate or its mixture of at least 2 kinds.
In substratum of the present invention, described inorganic nitrogen compound comprises urea, nitrite, nitrate, inorganic ammonium salt or its mixture of at least 2 kinds.
Substratum of the present invention can comprise: glucose 10-200g/L, Sodium Glutamate 1-20g/L, ammonium sulfate 0.5-5g/L, yeast extract paste concentration is the 1/2-1/10 of carbon source concentration, 5-20g/L preferably, described inorganic salt are take sodium salt as standard, 0.1-35g/L, VITMAIN B1 concentration 10-30ppm in the described trace element, vitamin B6 concentration 5-15ppm, vitamin B12 concentration 1-5ppm, vitamin H concentration 1-5ppm, 6-BA concentration 3-15ppm.
The pH of substratum of the present invention can be 4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5 or 9.0.
Substratum Glutamic Acid sodium content of the present invention can be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 10g/L, 11g/L, 12g/L, 13g/L, 14g/L, 15g/L, 16g/L, 17g/L, 18g/L, 19g/L or 20g/L.
The content of ammonium sulfate can be 0.5g/L, 1.0g/L, 1.5g/L, 2.0g/L, 2.5g/L, 3.0g/L, 3.5g/L, 4.0g/L, 4.5g/L or 5.0g/L in the substratum of the present invention.
The content of glucose can be 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L or 200g/L in the substratum of the present invention.
The content of yeast extract paste can be 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L or 100g/L in the substratum of the present invention.
The content of inorganic salt in the substratum of the present invention (take sodium salt as standard) can be 0.1g/L, 0.5g/L, 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L or 35g/L.
The concentration of VITMAIN B1 can be 10ppm, 15ppm, 20ppm, 25ppm or 30ppm in the substratum of the present invention.
The concentration of vitamin B6 can be 5ppm, 8ppm, 10ppm, 12ppm or 15ppm in the substratum of the present invention.
The concentration of vitamin B12 can be 1ppm, 2ppm, 3ppm, 4ppm or 5ppm in the substratum of the present invention.
The concentration of vitamin H can be 1ppm, 2ppm, 3ppm, 4ppm or 5ppm in the substratum of the present invention.
The concentration of VITAMIN 6-BA can be 3ppm, 6ppm, 9ppm, 12ppm or 15ppm in the substratum of the present invention.
In yet another aspect, the invention provides a kind ofly at method for culturing microbes, described method comprises during the fermentation and to adopt such as the described substratum of first aspect.
In cultural method of the present invention, described microorganism is little algae.
Cultural method of the present invention can be included in shaking flask preculture, first order seed cultivation, secondary seed cultivation and/or the three grade fermemtation cultivation and adopt the described substratum of first aspect.
Cultural method of the present invention can comprise:
A) activation culture adopts inclined-plane or plate streaking with the algae kind, and in 25 ℃ of constant temperature culture 3-5 days, full, the eugonic algae of picking form fell, and again carries out inclined-plane or plate streaking, in 25 ℃ of constant temperature culture 3-5 days;
B) shaking flask preculture, the algae of picking activation falls to being inoculated in the clean shaking flask, and 25 ℃, 150-300rpm, preferred 150-250rpm, most preferably shaking culture under the 200rpm;
C) fermentation amplification culture, the algae kind access first class seed pot that wherein the shaking flask preculture is obtained, inoculum size 2%-10%, preferred 4%-8%, most preferably 6%, pass into sterile air and stir, air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, most preferably 0.7vvm, stirring velocity 100-300rpm, preferred 150-250rpm, most preferably 200rpm, the control culture temperature is at 24-28 ℃, preferred 25-27 ℃, most preferably 26 ℃, carry out first order seed and cultivate; Fermentation seed liquid in the first class seed pot is seeded to the secondary seed tank, inoculum size 5%-15%, preferred 7%-13%, most preferably 10%, pass into sterile air and stir air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, 0.7vvm most preferably, stirring velocity 100-300rpm, preferred 150-250rpm, most preferably 200rpm controls culture temperature at 24-28 ℃, preferred 25-27 ℃, most preferably 25 ℃, carry out secondary seed and cultivate; Will be in the secondary seed tank carry out fermentation culture in the fermentation seed liquid inoculation three grade fermemtation tank, the preferred 7%-13% of inoculum size, most preferably 10%, lead to continuously in the process and add sterile air, air flow 0.4-1.0vvm, air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, 0.7vvm most preferably, stirring velocity 100-200rpm, preferred 120-180rpm, most preferably 150rpm controls culture temperature at 24-28 ℃, preferred 25-27 ℃, most preferably 26 ℃, carry out fermentation culture;
Wherein step b) and c) in the substratum that adopts be such as the described substratum of first aspect.Beneficial effect of the present invention:
(1) the present invention forms design by fermention medium, adopt the nutritive ingredient combination to come efficient controlled fermentation process pH fluctuation, make pH be controlled at constant and optimum range in, reduce or reduced by peripheral equipment and replenished the operation steps that soda acid is regulated fermentation pH, and the microbiological contamination hidden danger of bringing thus, greatly improve fermenting process controllability and stability;
(2) ammonium salt of method employing of the present invention can be used as nitrogenous source Substitute For Partial yeast extract paste, realizes the decrement of high price nitrogenous source, can greatly reduce culture medium cost.
Description of drawings
Fig. 1 is the schematic flow sheet of cultural method of the present invention.
Embodiment
Further specify technical scheme of the present invention below by embodiment.
Embodiment 1
Adopt substratum of the present invention (concrete composition sees Table 1), carry out the 500L of algae kind, 6 cubes, 60 cubes three grades of amplification culture according to the following step, algae kind Yu Haiyang thraustochytriale (Thraustochytrids) class:
At first, the algae kind is activated, adopt inclined-plane or plate streaking, in 25 ℃ of constant temperature culture 3-5 days, full, the eugonic algae of picking form fell, and again carries out inclined-plane or plate streaking, as shaking flask preculture algae kind, store method adopts cryogenic freezing after 25 ℃ of constant temperature culture 3-5 days.
Secondly, the shaking flask preculture, the picking algae falls to being inoculated in to contain in the clean shaking flask of aseptic above-mentioned substratum, 25 ℃, shaking culture under the 150-300rpm, this step can comprise that 2-3 level volume amplifies preculture, finish seed pre-culture after, shake-flask seed is incorporated in the sterile chamber, and this step is the seed pre-culture stage.
The fermentation amplification culture again, comprise one-level, secondary and three grades of amplification fermentation culture, flame differential pressure inoculation method access first class seed pot is adopted in the shake-flask seed inoculation, inoculum size 2%-10% passes into sterile air and stirs air flow 0.4-1.0vvm, 100-300rpm, the control culture temperature optimum 25 ℃, is carried out first order seed and is cultivated at 24-28 ℃.First order seed adopts pressure differential method to be seeded to the secondary seed tank that contains above-mentioned aseptic culture medium fermentation seed liquid in the first class seed pot after being cultured to certain phase, inoculum size 5%-15%, after the secondary seed tank is finished inoculation, pass into sterile air and stirring, air flow 0.4-1.0vvm, 100-300rpm, the control culture temperature is at 24-28 ℃, optimum 25 ℃, carry out secondary seed and cultivate.Secondary seed is cultured to and adopts pressure differential method to be seeded in the three grade fermemtation tank that contains above-mentioned aseptic culture medium fermentation seed liquid in the secondary seed tank behind the certain phase to carry out fermentation culture, inoculum size 5-15%, lead to continuously in the process and add sterile air, air flow 0.4-1.0vvm, 100-200rpm, the control culture temperature optimum 25 ℃, is carried out fermentation culture at 24-28 ℃.
At last, when dry cell weight increases to plateau or cell and is not suitable for continuing the situation such as cultivation and occurs, stop fermentation culture, put tank, fermented liquid is stored in the storage tank, pass into whizzer and carry out the concentrated collection of fermented liquid, after obtaining concentrated solution, pass into and obtain product in the spray-drying tower.
The two is consumed in cellular process, and pH produces the impact that raises and reduce on fermentation, and effect is cancelled out each other, and realizes the stable and efficient control of fermenting process pH.
Table 1: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 30 | VITMAIN B1 | 30 |
| Yeast extract paste | 15 | B6 | 5 |
| Sodium Glutamate | 1 | B12 | 1 |
| Ammonium sulfate | 0.5 | Vitamin H | 1 |
| Sodium-chlor | 12 | 6-BA | 3 |
| Sal epsom | 3 | ||
| Magnesium chloride | 3 | ||
| Repone K | 1 | ||
| Calcium sulfate | 1 |
Detect dry cell weight, sugared concentration, pH, amino nitrogen in the fermenting process, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation, when carbon source concentration is lower than 1%, add 50% sugar soln to fermented liquid sugar concentration 1.5-2%.Fermenting process does not need to adopt the extra supplemental acid alkali lye of peripheral equipment to regulate pH, and the pH of substratum is basicly stable in 5.0-7.0 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 2
Adopt substratum of the present invention (concrete composition see Table 2), adopt with embodiment 1 in identical algae kind and method 500L, 6 cubes, 60 cubes three grades of amplification culture of carrying out the algae kind:
Table 2: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 80 | VITMAIN B1 | 10 |
| Yeast extract paste | 30 | B6 | 15 |
| Sodium Glutamate | 15 | B12 | 5 |
| Ammonium nitrate | 4 | Vitamin H | 5 |
| Sodium-chlor | 5 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 1 | ||
| Calcium sulfate | 1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 5.5-6.8 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 3
Adopt substratum of the present invention (concrete composition see Table 3), adopt with embodiment 1 in identical algae kind and method 500L, 6 cubes, 60 cubes three grades of amplification culture of carrying out the algae kind:
Table 3: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 100 | VITMAIN B1 | 10 |
| Yeast extract paste | 40 | B6 | 15 |
| Sodium Glutamate | 20 | B12 | 5 |
| Ammonium chloride | 3 | Vitamin H | 5 |
| Sodium-chlor | 5 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 0.5 | ||
| Calcium sulfate | 0.1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 4.5-6.0 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 4
Adopt substratum of the present invention (concrete composition see Table 4), adopt with embodiment 1 in identical algae kind and method 500L, 6 cubes, 60 cubes three grades of amplification culture of carrying out the algae kind:
Table 4: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 200 | VITMAIN B1 | 10 |
| Yeast extract paste | 100 | B6 | 15 |
| Salt of wormwood | 20 | B12 | 5 |
| Ammonium nitrate | 5 | Vitamin H | 5 |
| Sodium-chlor | 12 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 1 | ||
| Calcium sulfate | 1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 5.3-6.5 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 5
Adopt substratum of the present invention (concrete composition sees Table 5), adopt grid algaes (Scenedesmus sp.) algae kind to carry out 500L, 6 cubes, 60 cubes three grades of amplification culture:
Table 5: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 60 | VITMAIN B1 | 20 |
| Yeast extract paste | 20 | B6 | 15 |
| Sodium glycocollate | 10 | B12 | 5 |
| Ammonium sulfate | 3.5 | Vitamin H | 5 |
| Sodium-chlor | 5 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 1 | ||
| Calcium sulfate | 1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 6.5-8.2 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 6
Adopt substratum of the present invention (concrete composition see Table 6), adopt with embodiment 5 in identical algae kind carry out 500L, 6 cubes, 60 cubes three grades of amplification culture:
Table 6: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 150 | VITMAIN B1 | 20 |
| Yeast extract paste | 70 | B6 | 15 |
| Sodium histidinate | 5 | B12 | 5 |
| Ammonium sulfate | 4.5 | Vitamin H | 5 |
| Sodium-chlor | 5 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 1 |
| Calcium sulfate | 1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 7.8-9.0 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Embodiment 7
Adopt substratum of the present invention (concrete composition see Table 7), adopt with embodiment 5 in identical algae kind carry out 500L, 6 cubes, 60 cubes three grades of amplification culture:
Table 7: substratum forms
| Component | Content (g/L) | Component | Content (ppm) |
| Glucose | 10 | VITMAIN B1 | 20 |
| Yeast extract paste | 5 | B6 | 15 |
| Sodium L-tyrosinate | 10 | B12 | 5 |
| Ammonium sulfate | 3.5 | Vitamin H | 5 |
| Sodium-chlor | 5 | 6-BA | 3 |
| Sal epsom | 1 | ||
| Magnesium chloride | 1 | ||
| Repone K | 1 | ||
| Calcium sulfate | 1 |
With similar among the embodiment 1, detect during the fermentation dry cell weight, sugared concentration, pH, amino nitrogen, add the nutritive elements such as carbon source, nitrogenous source according to the Growth of Cells situation.The pH of substratum is basicly stable in 6.8-8.3 in the fermenting process after testing, belongs to the suitable pH of the cultivation scope of marine alga.
Applicant's statement, the present invention illustrates detailed features of the present invention and method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method, does not mean that namely the present invention must rely on above-mentioned detailed features and method could be implemented.The person of ordinary skill in the field should understand, any improvement in the present invention to the increase of the equivalence replacement of the selected component of the present invention and auxiliary component, the selection of concrete mode etc., all drops within protection scope of the present invention and the open scope.
Claims (13)
1. substratum, comprise the combination of strong acid weak base salt and weak acid strong alkali salt in the described substratum, wherein, described strong acid weak base salt is organic and the inorganic acid ammonium salt, be preferably ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium oxalate or at least two kinds mixture wherein more preferably are ammonium sulfate, and described weak acid strong alkali salt is carbonic acid or amino acid whose sodium salt, sylvite or calcium salt or at least two kinds mixture wherein.
2. substratum according to claim 1 is characterized in that, the combination of described strong acid weak base salt and weak acid strong alkali salt is controlled to be 4.0-9.0 with the pH of described substratum, preferably 5.0-8.0, most preferably 6.0-7.0.
3. substratum according to claim 1 and 2, it is characterized in that described amino acid whose sodium salt, sylvite or calcium salt are sodium salt, sylvite or calcium salt or its mixture of at least 2 kinds of L-glutamic acid, aspartic acid, Methionin, arginine, Histidine, glycine, Serine, Threonine, halfcystine, tyrosine, L-Ala, α-amino-isovaleric acid, leucine, Isoleucine, proline(Pro), phenylalanine, tryptophane or methionine(Met).
4. substratum according to claim 1 and 2 is characterized in that, described weak acid strong alkali salt is Sodium Glutamate.
5. substratum according to claim 4 is characterized in that, the content of described substratum Glutamic Acid sodium is 1-20g/L, is preferably 5-15g/L, and more preferably 8-12g/L most preferably is 10g/L.
6. each described substratum is characterized in that according to claim 1-5, and the content of ammonium sulfate is 0.5-5g/L, is preferably 1-4g/L, and more preferably 2-3.5g/L most preferably is 3g/L.
7. each described substratum is characterized in that according to claim 1-6, also comprises other nitrogenous sources, carbon source, inorganic salt and trace element in the described substratum;
Preferably, described carbon source is waste molasses, sugar cane juice, Semen Maydis powder, sucrose, fructose, glucose, Zulkovsky starch, carbonic acid gas or its mixture of at least 2 kinds;
Preferably, described nitrogenous source is organic nitrogen compound, inorganic nitrogen compound or its mixture;
Preferably, described inorganic salt comprise sodium salt, magnesium salts, sylvite, molysite, calcium salt, vitriol, carbonate, supercarbonate, acetate or its mixture of at least 2 kinds; And/or,
Preferably, described trace element comprises VITMAIN B1, vitamin B6, vitamin B12, vitamin H, 6-benzyl aminoadenine (6-BA) or its mixture of at least 2 kinds.
8. substratum according to claim 7 is characterized in that, described organic nitrogen compound comprises yeast extract, yeast extract paste, corn steep liquor, peptone, amino acid, Sodium Glutamate or its mixture of at least 2 kinds; And/or,
Described inorganic nitrogen compound comprises urea, nitrite, nitrate, inorganic ammonium salt or its mixture of at least 2 kinds.
9. substratum according to claim 1 is characterized in that described substratum comprises: glucose 10-200g/L, Sodium Glutamate 1-20g/L, ammonium sulfate 0.5-5g/L, yeast extract paste concentration is the 1/2-1/10 of carbon source concentration, and described inorganic salt are take sodium salt as standard, 0.1-35g/L, VITMAIN B1 concentration 10-30ppm in the described trace element, vitamin B6 concentration 5-15ppm, vitamin B12 concentration 1-5ppm, vitamin H concentration 1-5ppm, 6-BA concentration 3-15ppm.
10. a method for culturing microbes is characterized in that, described method comprises that employing is such as each described substratum among the claim 1-9 during the fermentation.
11. method as claimed in claim 10 is characterized in that, described microorganism is little algae.
12., it is characterized in that described method is included in shaking flask preculture, first order seed cultivation, secondary seed cultivation and/or the three grade fermemtation cultivation and adopts such as each described substratum among the claim 1-9 such as claim 10 or 11 described methods.
13. method as claimed in claim 12, described method comprises:
A) activation culture adopts inclined-plane or plate streaking with the algae kind, and in 25 ℃ of constant temperature culture 3-5 days, full, the eugonic algae of picking form fell, and again carries out inclined-plane or plate streaking, in 25 ℃ of constant temperature culture 3-5 days;
B) shaking flask preculture, the algae of picking activation falls to being inoculated in the clean shaking flask, and 25 ℃, 150-300rpm, preferred 150-250rpm, most preferably shaking culture under the 200rpm;
C) fermentation amplification culture, the algae kind access first class seed pot that wherein the shaking flask preculture is obtained, inoculum size 2%-10%, preferred 4%-8%, most preferably 6%, pass into sterile air and stir, air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, most preferably 0.7vvm, stirring velocity 100-300rpm, preferred 150-250rpm, most preferably 200rpm, the control culture temperature is at 24-28 ℃, preferred 25-27 ℃, most preferably 26 ℃, carry out first order seed and cultivate; Fermentation seed liquid in the first class seed pot is seeded to the secondary seed tank, inoculum size 5%-15%, preferred 7%-13%, most preferably 10%, pass into sterile air and stir air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, 0.7vvm most preferably, stirring velocity 100-300rpm, preferred 150-250rpm, most preferably 200rpm controls culture temperature at 24-28 ℃, preferred 25-27 ℃, most preferably 25 ℃, carry out secondary seed and cultivate; Will be in the secondary seed tank carry out fermentation culture in the fermentation seed liquid inoculation three grade fermemtation tank, the preferred 7%-13% of inoculum size, most preferably 10%, lead to continuously in the process and add sterile air, air flow 0.4-1.0vvm, air flow 0.4-1.0vvm, preferred 0.5-0.8vvm, 0.7vvm most preferably, stirring velocity 100-200rpm, preferred 120-180rpm, most preferably 150rpm controls culture temperature at 24-28 ℃, preferred 25-27 ℃, most preferably 26 ℃, carry out fermentation culture;
Wherein step b) and c) in the substratum that adopts be such as each described substratum among the claim 1-9.
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| US20140093922A1 (en) | 2014-04-03 |
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